Acute lung injury (ALI) is a syndrome with significant morbidity and mortality, but its genetic susceptibility is not clearly understood. In the present study, we characterized functional promoter single nucleotide polymorphisms (SNPs) in the phase II antioxidant gene NQO1 (NAD(P)H:quinone oxidoreductase1) to evaluate its role in susceptibility to ALI. Three previously uncharacterized SNPs in the NQO1 promoter were selected for investigation. Luciferase assays were performed using constructs of each promoter polymorphism to evaluate function. Functional SNPs were genotyped in a prospective cohort of major trauma patients (N= 264) and assessed for association with development of ALI. The A/C SNP at −1221 decreased in vitro transcription of NQO1 at baseline and after exposure to hyperoxia and other oxidant stressors. Patients heterozygous for the −1221 C allele were at significantly lesser risk of ALI after major trauma compared with patients with wild-type alleles, even after adjustment for APACHE III score, and mechanism of trauma [OR, 0.46 (95% CI 0.23, 0.90); P= 0.024]. This study demonstrated that the AC genotype at position −1221 in the NQO1 gene caused decreased transcription and was associated with a lower incidence of ALI following major trauma. These novel findings may have important implications in diseases with oxidant stress aetiologies.